The primary function of the epidermis is to produce the protective, water-impermeable stratum corneum which permits terrestrial life. Sphingolipids (ceramides) are a marker of epidermal differentiation and are essential to its structure and function. Little is known about ceramide metabolism in keratinocytes or the relationship between epidermal differentiation and ceramide metabolism. The overall goal of this project is to understand this relationship and to elucidate the mechanisms involved in ceramide transport and metabolism in keratinocytes. The first specific aim is to determine the effect of epidermal differentiation state on the activities of ceramide glucosyltransferase and -glucocerebrosidase, two key enzymes in glucosylceramide metabolism. Enzyme activities will be determined in calcium-modulated human keratinocyte cultures, and in organotypic mouse and human keratinocyte cultures during the differentiation process. The second aim is to determine the effect of inhibition of these enzymes on epidermal differentiation. Cultures will be grown in the presence of specific inhibitors and the effects on the ultrastructural morphology of the cultures evaluated. The third aim is to determine the mechanism of glucosylceramide transport in keratinocytes. A fluorescent ceramide will be delivered to cultures and the changes in its cellular distribution over time examined using confocal microscopy. Biosynthetic conversion to fluorescent products will be monitored. the effect of known inhibitors of glucosylceramide transport and metabolism will be determined. Ultrastructural autoradiography and lipid analysis of organotypic cultures radiolabelled with synthetic ceramide will be used to examine the subcellular disposition of glucosylceramide. The last aim is to improve the organotypic culture system. Although the current systems closely approximate epidermis, the longevity and barrier function of the cultures are not optimal. to simulate the in vivo situation, a continuous culture perfusion system will be used. Glucose, lactate, and acetate levels in the culture medium will be used to evaluate the adequacy of the perfusion and the longevity and barrier function of the cultures will be assessed. Basic knowledge about epidermal sphingolipid biochemistry has important biomedical applications. Stratum corneum barrier function and the normal desquamation of corneocytes is disturbed in a wide variety of common and uncommon skin diseases, but the mechanism by which this occurs is still unknown. Either secondary changes in, or as yet undiscovered primary genetic defects in, many of the cellular processes associated with ceramide metabolism are likely to be responsible for this disruption of normal epidermal differentiation. Understanding normal epidermal sphingolipid metabolism is vital to understanding the role that disturbances in this essential part of epidermal lipid metabolism may play in cutaneous disease and to attempting to devise therapies to counteract these disturbances. Ultimately, these studies will contribute to fundamental knowledge about the regulation of epidermal differentiation at the molecular level.